PDK-53 is an attenuated dengue 2 strain that is being developed as a candidate vector for a tetravalent dengue (DEN) vaccine. An attenuating mutation in the RNA of PDK-53 is C57U in the 5'-untranslated region (UTR). Our experiments indicate that this mutation decreases translational gene expression to about 60%, and we suggest that attenuation arises because of a new upstream open reading frame (uORF) that interferes with the normal synthesis of the DEN polyprotein by ribosomes. We believe that the insertion of a uORF could be a general strategy for designing attenuated strains of a positive strand RNA virus with utility for vaccine design. Because ribosomal behavior on 5'-UTRs of capped RNAs is in general well understood, selected point sequence changes should result in predictable alterations of translation efficiency. We will test this strategy with several designed mutations in the 5'-UTRs of DEN-2. If successful, we will test the wider utility with limited mutations in the 5'-UTRs of Sindbis, DEN-4 and West Nile viruses. Vaccines for all the above viruses except Sindbis are medically desirable. The effects of mutations will be tested in translational reporter assays and in virus amplification assays in mammalian and C6/36 mosquito cells. These experiments will test the applicability and generality of the uORF strategy. The stability of the DEN-2 mutations will be tested over multiple passages. A subsequent round of mutations with DEN-2 will test the ability to adjust attenuation levels by design, and test the incorporation of additional mutations aimed at protecting against reversion towards wild type levels of gene expression. We expect that experience with differently placed uORFs will help to devise guidelines for better design of such mutations. Our understanding will be buttressed by experiments investigating ribosomal behavior on DEN-2 5'-UTRs containing uORFs, and by determining to what extent attenuation is correlated with altered ribosome access to the viral ORF.